Ex Vivo Mimicking of Inflammation in Organoids Derived From Patients With Ulcerative Colitis

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Studies on the anti-tumoral activity of 3-substituted indoles and azaindoles in B cell malignancies

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018.Indole-3-carbinol (3-hydroxymethylindole; I3C) is a natural product found in broadly consumed plants of the Brassica genus, such as broccoli, cabbage, and cauliflower, which exhibits anti-tumor effects through poorly defined mechanisms. I3C can be orally administered and clinical trials have demonstrated that I3C is safe in humans. We have described that I3C efficiently induces apoptosis in cell lines derived from EBV-positive Burkitt’s lymphomas (EBV+BL) (Perez-Chacon et al, 2014 Pharmacol. Res. 89:46) and in leukemic cells from chronic lymphocytic leukemia (CLL) patients. Interestingly, I3C was able to induce cell death of CLL cells and to synergize with fludarabine even in cells from patients with refractory CLL (Perez-Chacon et al, 2016. Clin. Cancer Res 22:134). We have studied whether other 3-substituted indoles also have a deleterious effect on the viability of the EBV+ BL cell lines Raji and BL60.2, and of CLL cells. Our results show that 3-substituted indoles with either carboxylic, methylcarboxylic, aminocarboxylic, cyanomethyl, carboxaldehide, dimethylaminomethyl, methoxymethyl or carboxamide groups failed to have any effect on the viability of the EBV+BL and CLL cells at concentrations up to 200 μM. Interestingly, not even 3-hydroxyethylindole and indole-2-methanol showed any cytotoxic effect, thus underlying the key role of the hydroxylmethyl group at 3 position for the anti-tumoral activity of I3C. It is noteworthy that indole-3-ol was cytotoxic in some of the tested cell lines. However, while I3C cytotoxicity was prevented by the caspases inhibitor zVAD-fmk, thus indicating that I3C induces apoptosis, zVAD-fmk failed to prevent indole-3-ol-induced cell death, thus suggesting that I3C and indol-3-ol kill by different mechanisms. In addition, we have also demonstrated that 7-azaindole and 3-hydroxymethyl-7-azaindole lack of any deleterious effect on the viability of B cell malignancies under study. This result indicates that the replacement of C for N at 7-position of the indole ring abolishes its anti-tumor activity. Finally, we have also observed that C6-methylated-I3C was more potent than I3C in inducing cell death in both EBV+ BL cell lines and in CLL cells, thus suggesting that substitution(s) at the benzene-fused ring (positions 4, 5, and/or 6) might enhance the anti-tumor activity of I3C and open the possibility to develop new I3C derivatives with improved anti-tumor activity.Peer reviewe

Butyrate Does Not Protect Against Inflammation-induced Loss of Epithelial Barrier Function and Cytokine Production in Primary Cell Monolayers From Patients With Ulcerative Colitis

BACKGROUND AND AIMS: In vitro studies using immortalised cancer cell lines showed that butyrate has an overall positive effect on epithelial barrier integrity, but the physiological relevance of cancer cell lines is limited. We developed epithelial monolayers from human tissue samples of patients with ulcerative colitis [UC] to assess the effect of butyrate on epithelial barrier function. METHODS: A protocol to establish monolayers from primary epithelial cells of UC patients [n = 10] and non-UC controls [n = 10] was optimised. The monolayers were treated with 8 mM sodium butyrate ± tumour necrosis factor alpha [TNFα] and type II interferon [IFNγ] for 48 h. Changes in transepithelial electrical resistance were monitored. Barrier gene expression levels were measured. Inflammatory proteins in the supernatant of the cells were quantified with OLINK. RESULTS: We demonstrated that primary monolayer cultures can be grown within 1 week of culture with robust resistance values and polarised tight junction expression. Butyrate treatment of the cultures increased resistance but was detrimental in combination with TNFα and IFNγ. The combined treatment further induced even higher IL8 mRNA and inflammatory protein secretion than for the inflammatory mediators alone. The observed effects were similar in cultures from patients and non-UC controls, suggesting that there were no patient-specific responses responsible for these findings. CONCLUSIONS: We found that butyrate does not protect against inflammation-induced barrier dysfunction and even worsens its effects in primary epithelial monolayers of UC patients and controls. The basic mechanisms of butyrate should therefore be reconsidered in future studies, in particular in patients with active inflammation and pre-existing barrier defects as is known for UC.status: publishe

Human intestinal epithelium in a dish: Current models for research into gastrointestinal pathophysiology

Determining the exact pathogenesis of chronic gastrointestinal diseases remains difficult due to the complex in vivo environment. In this review we give an overview of the available epithelial cell culture systems developed to investigate pathophysiology of gastrointestinal diseases. Traditionally used two-dimensional (2D) immortalised (tumour) cell lines survive long-term, but are not genetically stable nor represent any human in particular. In contrast, primary cultures are patient unique, but short-lived. Three-dimensional (3D) organoid cultures resemble the crypt-villus domain and contain all cell lineages, are long-lived and genetically stable. Unfortunately, manipulation of the 3D organoid system is more challenging. Combining the 3D and 2D technologies may overcome limitations and offer the formation of monolayers on permeable membranes or flow-chambers. Determining the right model to use will depend on the pathology of interest and the focus of the research, defining which cell types need to be included in the model.status: publishe

High Acetate Concentration Protects Intestinal Barrier and Exerts Anti-Inflammatory Effects in Organoid-Derived Epithelial Monolayer Cultures from Patients with Ulcerative Colitis

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD

Expression Levels of 4 Genes in Colon Tissue Might Be Used to Predict Which Patients Will Enter Endoscopic Remission After Vedolizumab Therapy for Inflammatory Bowel Diseases

BACKGROUND & AIMS: We aimed to identify biomarkers that might be used to predict responses of patients with inflammatory bowel diseases (IBD) to vedolizumab therapy. METHODS: We obtained biopsies from inflamed colon of patients with IBD who began treatment with vedolizumab (n = 31) or tumor necrosis factor (TNF) antagonists (n = 20) and performed RNA-sequencing analyses. We compared gene expression patterns between patients who did and did not enter endoscopic remission (absence of ulcerations at month 6 for patients with Crohn's disease or Mayo endoscopic subscore ≤1 at week 14 for patients with ulcerative colitis) and performed pathway analysis and cell deconvolution for training (n = 20) and validation (n = 11) datasets. Colon biopsies were also analyzed by immunohistochemistry. We validated a baseline gene expression pattern associated with endoscopic remission after vedolizumab therapy using 3 independent datasets (n = 66). RESULTS: We identified significant differences in expression levels of 44 genes between patients who entered remission after vedolizumab and those who did not; we found significant increases in leukocyte migration in colon tissues from patients who did not enter remission (P < .006). Deconvolution methods identified a significant enrichment of monocytes (P = .005), M1-macrophages (P = .05), and CD4+ T cells (P = .008) in colon tissues from patients who did not enter remission, whereas colon tissues from patients in remission had higher numbers of naïve B cells before treatment (P = .05). Baseline expression levels of PIWIL1, MAATS1, RGS13, and DCHS2 identified patients who did vs did not enter remission with 80% accuracy in the training set and 100% accuracy in validation dataset 1. We validated these findings in the 3 independent datasets by microarray, RNA sequencing and quantitative PCR analysis (P = .003). Expression levels of these 4 genes did not associate with response to anti-TNF agents. We confirmed the presence of proteins encoded by mRNAs using immunohistochemistry. CONCLUSIONS: We identified 4 genes whose baseline expression levels in colon tissues of patients with IBD associate with endoscopic remission after vedolizumab, but not anti-TNF, treatment. We validated this signature in 4 independent datasets and also at the protein level. Studies of these genes might provide insights into the mechanisms of action of vedolizumab.status: publishe